Characterisation of samples is used in a wide variety of applications, such as for example in the field of biology, biotechnology, chemistry and for clinical and medical purposes. One increasingly popular class of samples that need to be characterised are samples containing DNA and/or RNA.
Although numerous analysis techniques for qualification and quantification of samples exists, only few analysis techniques are as simple to perform, fast and accurate as spectrophotometry. One example of spectrophotometry is UV-VIS absorbance spectroscopy. During such experiments, samples are irradiated with UV-VIS radiation of different wavelengths, the radiation remaining after passage through the sample is detected and the absorbance at different wavelengths is determined. As particular components will show a particular absorbance at particular wavelengths, such a particular absorbance profile can be used as a fingerprint which allows, upon comparison with reference spectra, to identify the components. When more complex samples are studied, the absorbance features in the spectrum can be significantly overlapping, rendering the interpretation of spectra substantially more difficult.
For obtaining the proper samples to be characterized with UV-VIS spectroscopy, currently, often different types of extraction methods are used. These DNA and/or RNA extraction methods use different reagents and in particular the final diluents may differ significantly in composition and/or buffering capacity from “standard” samples. The diluent is typically specifically optimized for optimal yield in an extraction process. These different reagents and diluents that are used for extracting the proper nucleic acid containing sample typically strongly influence the final conditions for these samples, resulting in usually unknown varying conditions. As these unknown varying conditions may strongly influence the characterisation results, it is required that these conditions are either taken into account or are compensated for. Especially if accurate quantification and spectral analysis is required at low levels of nucleic acids, taking into account these varying conditions is required, as in these cases the chemical condition, in which the measurement is done, has a significant impact on UV-VIS spectroscopic characterisation.
One solution that was exploited in the past was dilution of the sample with a suitable buffer for rendering more or less standard conditions. Nevertheless, this typically will introduce an additional step (reducing the convenience of the analysis and throughput thereof). More importantly this may introduce dilution errors.
In other solutions, the influence of the buffer conditions on the spectral characterisation were ignored, leading to inaccurate quantification of DNA and/or RNA content of samples, thereby rendering the technique at present a less trustworthy technique for characterizing DNA and/or RNA containing samples.